For future improvement in agricultural crops and agricultural development, it is very important to impart useful properties, which cannot be imparted by hybridization breeding, to plants by transferring foreign genes to the plant cells. Also, the exploitation of a gene recombination technique of plants is indispensable for achieving matter production using plants with carbon dioxide as a raw material, as a novel matter production method that substitutes for petroleum-dependent matter production.
However, a plant transformation method has been established for model plants for use in general research, but has not yet been established or is very difficult to be carried out for many crops. Various gene transfer methods for plant cells such as Agrobacterium method, particle gun method, and whisker method have been developed. The act of sending a gene into cells is theoretically possible in itself for any plant. The reason why the transformation method has not yet been established for many plants is that, for example, a tissue culture method has not been established. In a general transformation method, undifferentiated cells such as callus are prepared from a plant tissue, and a foreign gene is transferred thereto using Agrobacterium or a particle gun. Since the foreign gene contains a selective marker gene such as an antibiotic resistance gene, cells harboring the transferred gene are selected by growing the cells on a medium containing the antibiotic after the gene transfer. Then, the selected cells are allowed to grow on a medium containing various concentrations of plant hormones for redifferentiation (rooting and shoot formation) to prepare a transformed individual. However, an approach for this tissue culture has not been established for many crops. Therefore, it has been difficult to obtain a transformed individual even if a gene has been transferred to the plants.
The in planta transformation method is a method which involves directly transferring a gene to an untreated plant individual or tissue and obtaining a transformed individual by a method that utilizes the life cycle of the plant. This method does not require tissue culture and is therefore applicable to even crops for which the transformation method has not yet been established. In the case of this method, the target cells for gene transfer are cells that produce progeny, i.e., cells of shoot apical meristems, pollens, and ovules. Examples of the in planta transformation most used include the DIP infiltration method of Arabidopsis thaliana. This method is a very convenient method which involves transferring a gene to, mainly, the ovule by dipping a flower of Arabidopsis thaliana in a culture solution of Agrobacterium, and pollinating this ovule to obtain transformed seeds. However, because of the difficulty in procedures of dipping a flower of the plant in a culture solution of Agrobacterium, this method is limited to Arabidopsis thaliana. Thus, the conventional gene transfer methods are difficult to be exploited in the in planta transformation, because the methods are limited by subject plants, tissue sizes, etc.
A cell-penetrating peptide (CPP) is known to have the function of transporting a complex comprising the peptide and another substance (e.g., a protein or a nucleic acid) through biomembranes in mammalian and human cell lines. However, the use of CPP is limited to plant cells. This is because, unlike animal cells, plant cells have a double bottleneck, i.e., a cell wall and a cell membrane, against the internalization of the complex comprising CPP. It has been found that a polycationic peptide concentrates negatively charged DNA through ionic interaction and forms a complex available for gene delivery, and the complex is useful in gene transfer to animal cells (Patent Literature 1). Also, a case using the polycationic peptide in gene transfer to a plant protoplast has been reported (Patent Literature 2). However, this method employs a cell wall-free protoplast and is not satisfactory in terms of gene transfer efficiency for plant cells.